Development of an online SPE–LC–MS-based assay using endogenous substrate for investigation of soluble epoxide hydrolase (sEH) inhibitors

Soluble epoxide hydrolase (sEH) is a promising therapeutic target for the treatment of hypertension, pain, and inflammation-related diseases. In order to enable the development of sEH inhibitors (sEHIs), assays are needed for determination of their potency. Therefore, we developed a new method utilizing an epoxide of arachidonic acid (14(15)-EpETrE) as substrate. Incubation samples were directly injected without purification into an online solid phase extraction (SPE) liquid chromatography electrospray ionization tandem mass spectrometry (LC–ESI–MS–MS) setup allowing a total run time of only 108 s for a full gradient separation. Analytes were extracted from the matrix within 30 s by turbulent flow chromatography. Subsequently, a full gradient separation was carried out on a 50X2.1 mm RP-18 column filled with 1.7 μm core–shell particles. The analytes were detected with high sensitivity by ESI–MS–MS in SRM mode. The substrate 14(15)-EpETrE eluted at a stable retention time of 96 ± 1 s and its sEH hydrolysis product 14,15-DiHETrE at 63 ± 1 s with narrow peak width (full width at half maximum height: 1.5 ± 0.1 s). The analytical performance of the method was excellent, with a limit of detection of 2 fmol on column, a linear range of over three orders of magnitude, and a negligible carry-over of 0.1% for 14,15-DiHETrE. The enzyme assay was carried out in a 96-well plate format, and near perfect sigmoidal dose–response curves were obtained for 12 concentrations of each inhibitor in only 22 min, enabling precise determination of IC50 values. In contrast with other approaches, this method enables quantitative evaluation of potent sEHIs with picomolar potencies because only 33 pmol L−1 sEH were used in the reaction vessel. This was demonstrated by ranking ten compounds by their activity; in the fluorescence method all yielded IC50 ≤ 1 nmol L−1. Comparison of 13 inhibitors with IC50 values >1 nmol L−1 showed a good correlation with the fluorescence method (linear correlation coefficient 0.9, slope 0.95, Spearman’s rho 0.9). For individual compounds, however, up to eightfold differences in potencies between this and the fluorescence method were obtained. Therefore, enzyme assays using natural substrate, as described here, are indispensable for reliable determination of structure–activity relationships for sEH inhibition

Quantitative Detection of Weak D Antigen Variants in Blood Typing using SPR

Modern techniques for quantifying blood group antibody-antigen interactions are very limited, especially for weaker interactions which result from low antigen expression and/or partial expression of the antigen structure. Surface plasmon resonance (SPR) detection is often used to monitor and quantify bio-interactions. Previously, a regenerable, multi-fucntional platform for quantitative RBC phenotyping of normal antigen expression using SPR detection was reported. However, detection of weaker variants were not explored. Here, this sensitivity study used anti-human IgG antibodies immobilized to a gold sensor surface to two clinically important types of weaker D variants using SPR; weak D and partial D. Positive pre-sensitised cells bind to the anti-human IgG monolayer, and the response unit (RU) is reported (&gt;100 RU). Unbound negative cells are directly eluted (&lt;100 RU). Weak D cells were detected between a range of 180-580 RU, due to a lower expression of antigens. Partial D cells, category D VI, were also positively identified (352-1147 RU), similar to that of normal D antigens. The detection of two classes of weaker D variants was achieved for the first time using this fully regenerable SPR platform, opening up a new avenue to replace the current subjective and arbitrary methods for quantifying blood group antibody-antigen interactions.</p